1.Technical Field (Field of the Invention)
The present invention relates to a purified sulfite reductase. More specifically, the sulfite reductase of the present invention catalyzes the reduction of sulfites to sulfides, effectively recovers the protein of the denatured fish meat when used in the denatured fish, and increases the number of reactive sulfhydryl groups and gel strength of the denatured fish protein. The present invention also relates to a process for producing the purified sulfite reductase, and the method for recovering the proteins of the denatured fish by using said sulfite reductase in solution or powder form.
2. Prior Art
Sulfite reductases extensively existing in plants and microorganisms are the enzymes catalyzing the reduction of sulfites to sulfides in the final step of assimilatory sulfate reductions, that is the key step of cysteine biosyntheses. During the reduction of sulfites to sulfides, where 6 electrons are provided in the reaction as follows: SO32−+6e−+6H+→S2−+3H2O, reduced nicotinamide adenine dinucleotide phosphate (NADPH) acts as the electron donor in Escherichia coli, NADPH or reduced methyl viologen (MVH) acts as the electron donor in yeast, and other donors in other organisms or tissues.
Muscle proteins are mostly proteins of numerous types with various physiological functions, formed by the interactions of L-amino acids via peptide bonds, disulfide bonds, hydrogen bonds, ionic bonds and hydrophobic bonds, and by the actions via dipole-dipole moments. The “denaturation of proteins” refers to changes in the secondary, tertiary and quaternary three-dimensional structures of the proteins without destroying peptide bonds.
Because the structures of denatured proteins form random helix in unfolded states or aggregated states with the functional groups on or between peptide chains interacting thereby losing the original physical, chemical and biological characteristics. The reasons for the protein denaturation is generally considered as follows: the moisture in the muscle is frozen which makes salt concentration raised, and the environment pH value around the proteins changed, thereby resulting in the precipitation of protein molecules due to salting out and the aggregation of hydrophobic groups between or in molecules aggregate, and at the same time, new hydrogen bonds, disulfide bonds and ionic bonds are formed, which renders the proteins aggregated and in turn denatured. In addition, water surrounding the protein molecules form combined water with the functional groups such as —SH, —COOH, —NH and —CO of the protein molecules. In addition, due to temperature drop, water molecules with lower bonding strength form ice crystals first. At the same time, due to volume expansion, the conformation of protein molecules changes, the functional groups are exposed, new bonds are formed intermolecularly or intramolecularly, thereby causing protein molecules aggregated and denatured.
It is proven that the reason why the denaturation of freeze-stored fish proteins results in the formation of covalent bonds is that sulfhydryl groups are oxidized to disulfide groups (Jiang et al., J. Food Sci. 63:777–781, 1998, Jiang et al., J. Food Sci. 60:652–655, 1998). In denatured fish meat caused by freeze-storing, not only the phenomena such as softening of fish meat, running-off of tissue fluid, formation of spongy tissues formation occur, but also the emulsification property, water-holding capacity, gel-forming ability and viscoelasticity are all worse than those of fresh fish. The reason for these deteriorations is mainly because of the denaturation of myofibrillar proteins.
After being denatured, proteins form regular network structures, which make muscle proteins have characteristic of elasticity after being set by heat. Gelation of fish proteins, by which elasticity is given to the fish proteins, is one of the most important characteristics during processing fish proteins into refined products. The gelation of proteins is influenced by many factors, such as salt concentration, pH value, temperature, protein concentration, components, additives, ionic strength, freshness, and so on. It is known from the biochemical characteristics of the refined products that the bonds for forming network structures include hydrogen bonds, hydrophobic bonds, ionic bonds, disulfide bonds and other covalent bonds.
In addition, gel strength is an index to determine the quality of refined products of surimi. The elasticity of refined products is influenced by many factors, such as salt concentration, pH value, temperature, protein concentration, components, additives, ionic strength, freshness, and so on. Therefore, there have been proposed various methods to enhance the elasticity of the refined products of fish meat.
The gel-forming ability of fish meat decreases during freezing storage. The reason is mainly because of the gradual decrease in solubility of fish muscle proteins, which is considered resulting from the formation of polymers such as dimers and multimers of myosins due to the formation of disulfide and other covalent bondings. The formation of disulfide bonds among muscle proteins results in the aggregation and denaturation of actomyosin of fish muscle.
As described above, the gel-forming ability of fish meat decreases during freezing storage that causes the fish meat unsuitable for the processing of protein colloidal foods. The applications of freeze-stored fish meat in the production of protein colloidal foods are thus limited.
Although an alkaline washing treatment has long been employed to improve the gel-forming ability of mackerel surimi, it does not benefit color improvement. Although increase in alkaline washing cycles or using ozonic bleaching could substantially improve the color of mackerel, it results in deteriorations of gel-forming properties of the mackerel muscle proteins.
In addition, as for frozen surimi obtained by adding chemical reducing agents such as cysteine, sodium disulfite, ascorbic acid, and so on, into denatured fish muscles, the total sulfhydryl groups and reactive sulfhydryl groups of actomyosins, and the amount of extractable actomyosins are all much higher than those of frozen surimi to which the reducing agents are not added. Although the gel-forming ability of proteins decreases due to freezing, the addition of reducing agents could recover most of the proteins. Therefore, it is shown that reducing agents could recover the aggregated and denatured actomyosins. The addition of reducing agents results in reducing disulfide bonds of frozen denatured fish meat to sulfhydryl bonds during grinding. The disulfide bonds are re-formed during gelation of refined products whereby the network structures become more firm and stable. However, the chemical reducing agents are considered as external additives and are not easy to accept by the consumers.
The inventors of the present invention proposed to use sulfite reductases derived from microorganisms to replace those chemical reducing agents. For instance, Jiang et al. investigated the effect of sulfite reductases from yeasts on recovering denatured fish muscle protein of ozonically decolored/denatured and frozen denatured mackerel surimi, and found that the crude sulfite reductase could recover the denatured muscle proteins and enhance the gel-forming ability of refined products (Jiang et al., J. Food Sci. 60: 652–655, 1998; Jiang et al., J. Food Sci. 60: 777–781, 1998). Furthermore, Wu et al. applied crude lyophilized powders of the sulfite reductase prepared from Saccharomyces cerevisiae, applied to the processing of frozen fish, and also found that the gel-forming ability of refined products were substantially enhanced (Wu et al., J. Food Sci. 65: 1400–1403, 2000). However, sulfite reductases from microorganisms have never been prepared, the effects thereof on the recovery of denatured fish muscles have never been investigated, either.